Receptor tyrosine kinases (RTKs) are key regulators of intercellular communication that controls cell growth, proliferation, differentiation, survival and metabolism. About 20 different RTK families have been identified that share a similar structure, namely an extracellular binding site for ligands, a transmembrane region and an intracellular tyrosine kinase domain (1). Extracellular ligand binding induces or stabilizes receptor dimerization leading to increased RTK kinase activity. The intracellular catalytic domain displays the highest level of conservation among RTKs and includes the ATP-binding site that catalyzes receptor autophosphorylation of cytoplasmic tyrosine residues, which serve as docking sites for Src homology 2 (SH2)-and phosphotyrosine-binding (PTB) domain-containing proteins such as Grb2, Shc, Src, Cb1 or phospholipase C γ. These proteins subsequently recruit additional effectors containing SH2, SH3, PTB and pleckstrin-homology (PH) domains to the activated receptor, which results in the assembly of signaling complexes at the membrane and the activation of a cascade of intracellular biochemical signals.
The most important downstream signaling cascades activated by RTKs include the Ras-extracellular regulated kinase (ERK)-mitogen activated (MAP) kinase pathway, the phosphoinositide 3-kinase (PI 3-kinase)-Akt and the JAK/STAT pathway. The complex signaling network triggered by RTKs eventually leads either to activation or repression of various subsets of genes and thus defines the biological response to a given signal.
The activity of RTKs and their mediated cellular signaling is precisely coordinated and tightly controlled in normal cells. Deregulation of the RTK signaling system, either by stimulation through growth factor and/or through genetic alteration, result in deregulated tyrosine kinase activity. These aberrations generally result in RTKs with constitutive or strongly enhanced kinase activity and subsequent signaling capacity, which leads to malignant transformation. Therefore, they are frequently linked to human cancer and also to other hyperproliferative diseases such as psoriasis (2). The most important mechanisms leading to constitutive RTK signaling include overexpression and/or gene amplification of RTKs, genetic alterations such as deletions and mutations within the extracellular domain as well as alterations of the catalytic site, or autocrine-paracrine stimulation through aberrant growth factor loops.
For example, in many human cancers, gene amplification and/or overexpression of RTKs occurs, which might increase the response of cancer cells to normal growth factor levels. Additionally, overexpression of a specific RTK on the cell surface increases the incidence of receptor dimerization even in the absence of an activating ligand. In many cases this results in constitutive activation of the RTK leading to aberrant and uncontrolled cell proliferation and tumor formation. An important example for such a scenario is HER2, also known as ErbB2, that belongs to the epidermal growth factor (EGF) receptor family of RTKs. Overexpression of HER2 was found in various types of human cancers, especially in human breast and ovarian carcinomas (3). Most importantly, aberrantly elevated levels of HER2 correlate with more aggressive progression of disease and reduced patient survival time (4). EGFR, which was the first receptor tyrosine kinase to be molecularly cloned (5), also plays a fundamental role in tumorigenesis. EGFR is frequently overexpressed in non-small-cell lung, bladder, cervical, ovarian, kidney and pancreatic cancer and in squamous-cell carcinomas of the head and neck (6). The predominant mechanism leading to EGFR overexpression is gene amplification with up to 60 copies per cell reported in certain tumors (7). In general, elevated levels of EGFR expression are associated with high metastatic rate and increased tumor proliferation (8).
Since tyrosine kinases have been implicated in a variety of cancer indications, RTKs and the activated signaling cascades represent promising areas for the development of target-selective anticancer drugs. One approach to inhibit aberrant RTK signaling is the development of small-molecule drugs that selectively interfere with their intrinsic tyrosine kinase activity and thereby block receptor autophosphorylation and activation of downstream signal transducers (9).
Several methods have been developed to screen compound libraries in order to identify RTK-specific inhibitors, most of which utilize biochemical assays (10). One important aspect to consider for the selection of effective tyrosine kinase inhibitors is that these compounds must be able to permeate through cellular membranes and function in an intracellular environment for the necessary period of time. In addition, in order to become potential drug candidates kinase inhibitors must not show cytotoxic effects. It is therefore desirable to have a cellular system for the primary screening of compounds capable of inhibiting RTK activity. The requirements for such in vivo assays are the ability to examine a specific cellular process triggered by a defined target and a means to readily measure its output in a high-throughput screening system (HTS). The availability of an increasing number of biotechnological tools to genetically modify cells and microorganisms have allowed the development of simple read-out assays for cellular processes that can be readily applied to automated systems in HTS (11-14). Cellular screens should ideally be performed with cells of human origin, which evidently provide the most physiologically relevant model system. However, the effects of redundant processes on the measured output can be difficult to control and to distinguish from the effects that are expected to be specific for the defined target; and genetic manipulation of mammalian cells is generally problematic and time-consuming. Moreover, human cells are expensive to culture and sometimes difficult to propagate in automated systems used for HTS. Microorganisms such as yeast present a convenient alternative for measuring the activity of defined human proteins in a heterologous, yet cellular (eukaryotic) environment. In yeast cells, the function of human proteins can often be reconstituted and aspects of some human physiological processes can be recapitulated because of the high degree of conservation of basic molecular and cellular mechanisms between yeast and human cells (14-17). The fact that many human proteins function in yeast indicates that the required conformation, stability, protein-protein interaction, etc. are taking place in this eukaryotic organism.
Although there exist already methods for the isolation of receptor tyrosine kinase inhibitors, there is a need for a reliable cell-based method for the identification and/or validation of inhibitors of a receptor tyrosine kinase that permeate cell membranes and that are not cytotoxic.